Toxic effect on the rat small intestine of chronic administration of asbestos in drinking water

Toxicology Letters, 39 (1987) 205-209

205

Elsevier

TXL 01890

TOXIC EFFECT ON THE RAT SMALL INTESTINE OF CHRONIC ADMINISTRATION OF ASBESTOS IN DRINKING WATER (Asbestos

treatment;

THOMAS

intestinal

J. DELAHUNTY

permeability

and DANIEL

defect)

HOLLANDER

Division of Gastroenterology, Department of Medicine, University of California, Irvine, CA (U.S.A.) (Received

29 December

(Accepted

11 August

1986) 1987)

SUMMARY Sprague-Dawley imately 7 mg/day

rats were given a 0.5 g/l Chrysotile asbestos solution in their drinking water (approxingested) for 1.5 years and compared to control rats of the same age. During this time

there were no differences maintained

in weight or appearance

under the same conditions.

formed

using

recovery

of lactulose

a gavage/urinary

of the controls

(1.01

However,

collection

rats in comparison permeability

technique,

in the urine of asbestos-treated + 0.08, P
of the asbestos-treated when in viva intestinal some

significant

changes

rats was 0.66 + 0.07%,

The recovery

of mannitol

were

significantly

was similarly

to controls

studies were pernoted.

The

less than that

decreased

(2.2kO.28

vs. 3.0 + 0.17, P< 0.02), but that of rhamnose was unchanged. Creatinine clearance studies indicated that there was no impairment of kidney function in the asbestos-treated group and polarized light microscopy did not reveal any asbestos

fibers in sections

posure

of rats to asbestos

fibers in the drinking

of the small bowel. The results suggest

absorb

some non-metabolizable

water

results

in a decreased

that the chronic

ability

of the intestine

exto

sugars.

INTRODUCTION

Asbestos has a wide variety of commercial applications because of its unique combination of properties such as fire resistance, inertness, heat resistance, fibrous nature and availability [l]. Thus, apart from the obvious occupational exposure of

Address

for correspondence:

Department Abbreviation:

0378-4274/87/$

of Medicine, HPLC,

03.50

Thomas University

high pressure

0

Delahunty,

Ph.D.,

Division

of California,

Irvine,

CA 92717,

of Gastroenterology, U.S.A.

liquid chromatography.

1987 Elsevier

Science

Publishers

B.V. (Biomedical

Division)

Rm.

C340,

206

individuals engaged in asbestos manufacture, large segments of the general population can be exposed to asbestos fibers through contaminated air, food or water /2,3]. For example, up to 30 x LO5fibers/l have been detected in the drinking water of certain cities [4]. Although the predisposition of workers in the asbestos industry to developing mesotheliomas and pleuro-pulmonary neoplasia is by now well established [5], an enhanced risk of cancer development from a high fiber content in the drinking water is less apparent. [6], Because of the long incubation time required for the development of asbestos-related tumors [7], and since access to asbestos-exposed human populations is limited, we elected to look for physiological alterations which asbestos fibers might cause in the rat intestine, on the premise that intestinal permeability changes could be an indication of altered ep~the~~a1cell function and subsequent susceptibility to carcinogenic agents. MATERIALS

AND METHODS

Asbestos administration We used male Sprague-Dawley rats (initial weight: 150-200 g) which had free access to Purina Rodent Chow during the entire experiment. The asbestos-treated group was given a 0.5 g/l asbestos solution prepared in the following manner: 1 g of agar powder (Sigma Chemical Co., St. Louis, MO) was placed in 1 liter of hot water. This solution was mixed and kept at approximately 85°C for t h until the solution turned from a cloudy to clear suspension, After cooling, OS g/l of Chrysotife asbestos (Bureau of Mines, Washington, DC) was added and mixed for another hour until a final uniform white suspension was formed. This solution was stored at 3°C for up to 3 weeks. After 1.5 years of treatment, the mean weight of the asbestos-treated and control rats was 353 g and 368 g, respectively. Intestinal permeability assay The anmals were gavaged with 3 ml of solution consisting of 5 g/d1 of rhamnose, mannitol and lactulose. After collection for 6 h in specially constructed chambers, the urine was alka~~n~~edby the addition of 0.5 ~1 of saturated NaOH to a 5 ml aliquot. This was left standing for I5 mm and centrifuged for IO min. The supernatant was mixed with 2 g of washed ion-exchange resin ~Amberlite MB-3), vortexed for 10 s and recentrifuged. The clear supernatant was then removed and stored at - 2O”C, pending analysis. HPL C analysis A 20 ~1 aliquot of the clear urine extract was injected into an HPLC instrument equipped with a 20 ~1 loop, a polymeric guard column and a CHO-620 cation exchange column at 85°C (Interaction Chemicals, Mountain View, CA). We used an LKB dual piston HPLC pump (model 2150), an LKB refractive index detector

207

(LKB, Bromma,

Sweden),

and a Shimadzu

MO). Concentrations

of unknowns

tained with standard published [S].

concentrations

Integrator

were calculated of the sugars.

(Shimadzu referring

Corp.,

St. Louis,

to the peak areas ob-

This method

has already

been

Histological examination When the permeability studies were completed, the intestines were promptly removed from each animal after ether anesthesia, rinsed in 0.15 mol/l NaCl, and the entire mucosal surface examined macroscopically for obvious lesions. Multiple representative sections of the small and large intestines of asbestos-treated and untreated animals were processed for light microscopy. The tissue was fixed in 10% buffered formaldehyde and embedded in paraffin. 5 pm sections were cut, stained with hematoxylin-eosin and assessed for any abnormalities.

Creatinine clearance studies All animals were assessed for creatinine clearance by collecting the 24 h urinary output and approximately 1 ml of blood from the tail vein. The creatinine concentrations were measured by the Jaffe reaction as modified by Heinegard et al. [9] using reagents supplied by Sigma. RESULTS

The percent recoveries of the 3 non-metabolizable sugars in the urines of the asbestos-treated and control rats can be seen in Table I. The recovery of lactulose was reduced by 3.5% in comparison to that of control rats (P
I

TABLE PERCENT Values

URINARY

expressed

RECOVERY

are the mean

OF 3 COMPOUNDS

+ SD. Each animal

Controls

WITH

was studied

ASBESTOS

TREATMENT

3 times.

Treated

P

Change

Lactulose

1.01 + 0.08

0.66

+ 0.06

<0.005

Rhamnose

5.83

f

0.16

5.10

+ 0.70

NS

0

Mannitol ?I;1

3.00

t 0.17 3

2.20

+ 0.28 4

<0.02

27

‘Number

of animals

in each group.

35

(%)

208

pattern of recovery was similar with asbestos-treated rats. The creatinine clearances of the asbestos-treated and control rats were 94 + 12 and 95 f 11 pl/min, respectively. DISCUSSION

The studies reported here were an attempt to delineate changes in intestinal permeability which might result from the long-term exposure of rats to asbestos in the drinking water. Previous experiments by the authors showed that the permeability function of rat and guinea pig intestine could be monitored by the oral administration of non-metabolizable compounds such as polyethylene glycol, lactulose, rhamnose, mannitol, and azure A [ 10,111. The total urinary excretion of the probes was then estimated by analyzing the urine produced in 6 h using radiochemical and chromatographic techniques. With this method we obtained evidence that asymptomatic relatives of Crohn’s patients had an enhanced intestinal permeability of polyethylene glycol which suggested that susceptibility to the disease might be related to genetic factors [12]. We also showed that the administration of the inflammatory agent, carrageenan, in the drinking water resulted in an enhanced permeability of rat and guinea pig intestine to polyethylene glycol [lo]. A recent comparative study of humans, rats, guinea pigs and hamsters revealed that the guinea pig intestine was closest to the human when the permeability of lactulose, rhamnose and mannitol were assessed [l 11. The experiment described here was an attempt to delineate intestinal abnormalities which might be ascribed to the presence of asbestos in the drinking water. Our data showed that the intestinal penetration of lactulose and mannitol was significantly reduced in comparison to control animals, suggesting that the usual route for penetration of these compounds was partially blocked by the asbestos fibers. Since the asbestos has no significant effect on rhamnose uptake, it is likely that this sugar is at least partially absorbed by a route different from that of the other compounds. The finding that there was no difference in the clearance of creatinine suggests that the asbestos treatment did not cause impairment of kidney function which might have caused the observed decrease in the urinary recovery of lactulose or mannitol. At present we do not know how medium-sized permeability probes such as lactulose penetrate the intestinal barrier. Because of the beta linkage, lactulose is not appreciably hydrolyzed by intestinal enzymes [13], and therefore must traverse the intestinal wall intact. The small amount that penetrates to the serosal side most likely uses the paracellular route, since the disaccharide is not soluble in the lipid component of the cell membrane. Although the patency of this route is controlled by ‘tight’ junctions, electrolytes readily penetrate this barrier [14]. Thus, we can speculate that the asbestos fibers can penetrate these junctions and block the transport of lactulose. Electron microscopy of the tight junctions in the intestinal

209

sections will explore this possibility. The fact that we found no difference in rhamnose permeability probably reflects the fact that this monosaccharide is readily taken up and secreted by the mucosal cells along with any other monosaccharide present in the intestinal lumen. Since we found no evidence for asbestos fibers lodging within the cells, this particular route was probably not affected. Although mannitol readily diffuses across the intestinal barrier in humans, its rate of penetration in rats is much slower [ 111, possibly reflecting the fact that the paracellular route predominates in this species. ACKNOWLEDGEMENTS

The authors would like to thank the Goldsmith Foundation and the CRCC for supporting this research. The services of Dr. Thomas Ulich, Pathology Department, UC1 are also gratefully acknowledged. REFERENCES I

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